1. Technical Field
The present invention relates generally to the field of sophorolipids and more specifically to new compositions of matter for uses of sophorolipids as antimicrobial and antifungal agents; as biopesticides; for uses as drugs to treat HIV, septic shock, cancer, asthma, and dermatological conditions; as spermicidal agents; as anti-inflammatory drugs; as ingredients in cosmetics; as building blocks for monomers and polymers; and as self-assembled templates for further chemical elaboration.
2. Prior Art
Surfactants as cosmetics (neutral, anionic emulsifiers & surfactants); surfactants as antimicrobial and antifungal agents; surfactants as therapeutic agents.
Nature has evolved a class of compounds, known as sophorolipids that have antimicrobial properties. These natural biopesticides, which can be classified as microbial surfactants, are amphiphilic molecules produced by fermentation using substrates consisting of carbohydrates and lipids. Microbial biosurfactants are surface active compounds produced by various microorganisms. They lower surface and interfacial tension and form spherical micelles at and above their critical micelle concentration (CMC). Microbial biosurfactants generally have an amphiphilic structure, possessing a hydrophilic moiety, such as an amino acid, peptide, sugar or oligosaccharide, and a hydrophobic moiety including saturated or unsaturated lipid or fatty acids. Sophorolipids are produced by yeasts, such as Candida bombicola (see FIG. 1), Yarrowi alipolytica, Candida apicola, and Candida bogoriensis. 
They consist of a hydrophilic carbohydrate head, sophorose, and a hydrophobic fatty acid tail with generally 16 or 18 carbon atoms. Sophorose is an unusual disaccharide that consists of two glucose molecules linked β-1,2. Furthermore, sophorose in sophorolipids can be acetylated on the 6′- and/or 6″-positions (FIG. 1). One fatty acid hydroxylated at the terminal or subterminal (ω-1) positions is β-glycosidically linked to the sophorose molecule. The fatty acid carboxylic acid group is either free (acidic or open form) or internally esterified generally at the 4″-position (lactonic form) (FIG. 1). The hydroxy fatty acid component of sophorolipids generally has 16 or 18 carbon atoms with generally one unsaturated bond (Asmer et al. 1988; Davila et al. 1993). However, the sophorolipid fatty acid can also be fully saturated. As such, sophorolipids synthesized by C. bombicola consist of a mixture of molecules that are related. Differences between these molecules are found based on the fatty acid structure (degree of unsaturation, chain length, and position of hydroxylation), existing in the lactonic or ring-opened form, and the acetylation pattern. Sophorolipids derivatives disclosed herein are described based on the predominant fatty acid constituent, 17-hydroxyoleic acid, produced by C. bombicola when fed crude oleic acid as its fatty acid source. However, because changes in the lipid feed lead to different sophorolipids as described above, variations in feedstock also will result in changes in composition of modified sophorolipid structures that are disclosed herein.
Work has been carried out to “tailor” sophorolipid (SL) structure during in vivo formation. These studies have mainly involved the selective feeding of different lipophilic substrates. For example, changing the co-substrate from sunflower to canola oil resulted in a large increase (50% to 73%) of the lactonic portion of SLs. See Zhou, Q.-H., et al., J. Am. Oil Chem. Soc. 1995, 72, 67; Zhou, Q.-H., et al., J. Am. Oil Chem. Soc. 1992; 69, 89; Asmer, H. J., et al., J. Am. Oil; hem. Soc. 1988, 65, 1460; Davila, A. M., et al., Appl. Microbiol. Biotech. 1992, 38, 6; Tulloch A. P., et al., Can. J. Chem. 1962, 40, 1326. Also, unsaturated C-18 fatty acids of oleic acid may be transferred unchanged into sophorolipids. Rau, U., et al., Biotechnol. Lett. 1996, 18, 149. Finally, lactonic and acidic sophorolipids are synthesized in vivo from stearic acid with similar yields to oleic acid-derived sophorolipids. Felse, P. A., et al., Enzyme and Microbial Technology 2007, 40 316-323. Thus, to date, physiological variables during fermentations have provided routes to the variation of sophorolipid compositions.
As noted above, fermentation by different microorganisms, Candida bombicola, Yarrowi alipolytica, Candida apicola, and Candida bogoriensis, leads to sophorolipids of different structure noted above, the variations in sophorolipids based on fatty acid feedstocks and organisms leads to a wide array of sophorolipids including lactonic and acidic structures. An additional modification that is relevant to the acidic sophorolipids is the cleavage of the sophorose to the corresponding glucose-based glucolipids. Treatment of acidic sophorolipids with enzymes β-Glucuronidase (Helix pomatia), Cellulase (Penicillium funiculosum), Clara diastasea, Galactomannanase (Aspergillus niger), Hemicellulase (Aspergillus niger), Hesperidinase (Aspergillus niger), Inulinase (Aspergillus niger), Pectolyase (Aspergillus japonicus), or Naringinase (Penicillium decumbens) afford glucolipids over a range of pH values. Rau, U., et al., Biotechnology Letters 1999, 21, 973-977).
The chain length of the fatty acid carbon source fed can result in changes in the predominant fatty acid incorporated into the sophorolipid produced. For example, Davila et al. (1994) found that when hexadecane and octadecane were fed in fermentations, over 70% of the hydroxylated fatty acids found in sophorolipids were hexadecanoic and octadecanoic acids, respectively. When shorter alkanes such as tetradecane are fed as substrates in fermentations, only a minor fraction of the sophorolipids produced by the organism consist of the corresponding hydroxylated shorter chain fatty acid. Instead, the vast majority of these shorter chain fatty acids are elongated to either C16 or C18 fatty acids. Similarly, when longer alkanes such as eicosane (C20) are fed to the sophorolipid producing organism, generally longer chain fatty acids are metabolized via β-oxidation to shorter chain length hydroxylated C16 and C18 fatty acids.
Furthermore, the degree of lactonization of sophorolipids and acetylation of the sophorose polar head may be influenced by the carbon source used. For example, Davila et al. (1994) claims that sophorolipids produced from oils are formed with higher levels of diacetylated lactones than sophorolipids produced from the corresponding fatty acid ester feedstocks. Davila A. M., et al., Sophorose lipid production from lipidic precursors-Predictive evaluation of industrial substrates. J. Ind. Microbiol. 13:249-257 (1994).
It is known that by modification of sophorolipids, their physical properties can be manipulated (Zhang et al., 2004). Modifications of SLs were performed so that the chain length of the n-alkyl group (methyl, ethyl, propyl, butyl, hexyl) esterified to the sophorolipid fatty acid was varied. The effect of the n-alkyl ester chain length on interfacial properties of corresponding sophorolipid analogues was studied (FIG. 2).
The cmc and minimum surface tension have an inverse relationship with the alkyl ester chain length. That is, cmc decreased to ½ per additional CH2 group for the methyl, ethyl, and propyl series of chain lengths. These results were confirmed by fluorescence spectroscopy. Adsorption of sophorolipid alkyl esters on hydrophilic solids was also studied to explore the type of lateral associations. These surfactants were found to absorb on alumina but much less on silica. This adsorption behavior on hydrophilic solids is similar to that of sugar-based nonionic surfactants and unlike that of nonionic ethoxylated surfactants. Hydrogen bonding is proposed to be the primary driving force for adsorption of the sophorolipids on alumina. Increase in the n-alkyl ester chain length of sophorolipids caused a shift of the adsorption isotherms to lower concentrations. The magnitude of the shift corresponds to the change in cmc of these surfactants (FIG. 2). This study suggests that by careful modulation of sophorolipid structure via simple chemical modification, dramatic shifts in their surface-activity can be achieved to ‘tune’ their properties for a desired interfacial challenges. Furthermore, changes in interfacial properties are expected to relate to changes in biological properties.
Prior art describes limited ways in which sophorolipids can be modified for use as building blocks for polymer synthesis. For example, a mixture of sophorolipids produced by Torulopsis bombicola was esterified by reaction with sodium salts of n-alkanols. Using Novozym 435 as catalyst, the SL methyl ester, in the absence of acylating agent or with the acylating agent at a concentration less than equimolar, gave sophorolactone (Bisht et al, 1999). Spectral analysis of this compound showed that a synthetic analogue of microbially produced macrolactone was formed. Sophorolactone differs from the natural sophorolipid lactone in that the fatty acid carboxyl carbon is linked to the C-6 hydroxyl, not to the C-4 hydroxyl (FIG. 20). Subsequent acrylation of the new non-natural lactonic form of SL, catalyzed by Novozym 435, led to formation of the C-6 monoacryl derivative (Bisht et al, 2000). See FIG. 20.
The translation of complexity from natural sophorolipid building blocks to polymers was explored. In one example, a component of the natural sophorolipid mixture was used as a monomer for polymer synthesis. Specifically, lactonic sophorolipids, derived from fermentation of Candida bombicola, were polymerized by ring-opening metathesis polymerization (ROMP) catalysis. Poly(sophorolipid), poly(SL), was prepared in yield and Mn up to 89% and 103 000, respectively (FIG. 3). By this chemo-biocatalytic route, unique polymers with disaccharide, ester, and monounsaturated hydrocarbon moieties were prepared. The unique poly(SL) structure consists of C18 oleic-like aliphatic segments (90% cis-configured double bonds) that alternate with bulky diacetylated disaccharide moieties (Zini et al., 2008). Solid-state properties were investigated using TGA, DSC, TMDSC, and variable-temperature X-ray diffraction. Poly(SL) is a solid at room temperature that undergoes a glass transition at 61° C. and melts at 123° C. The crystal phase is associated with ordered packing of aliphatic chain segments. Semicrystalline poly (SL) also displays a long-range order (d=2.44 nm) involving sophorose groups that is found to persist after crystal phase melting (in high-T diffractograms) with a slightly shortened distance (2.27 nm). Upon annealing at 80° C., poly(SL) recrystallizes and, concomitantly, the disaccharide units space out again at 2.44 nm. An exothermal phenomenon that immediately follows melting and is revealed by TMDSC might be associated with the observed adjustment of sophorose units spacing in the melt. See FIG. 3.
Various sophorolipid (SL) structures can be used to create self-assembled scaffolds that can serve as templates for further chemical elaboration. For example, non-acetylated acidic sophorolipids are bola amphiphiles with unique structures that include an asymmetrical polar head size (disaccharide vs. COOH), a kinked hydrophobic core (cis-9-octadecenoic chain), and a non-amide polar-nonpolar linkage (Zhou et al, 2004). Light microscopy, small- and wide-angle X-ray scattering, FT-IR spectroscopy, and dynamic laser light scattering were used to investigate supramolecular structures of SL self-assembled aggregates at different pH values. In acidic conditions (pH<5.5), giant twisted and helical ribbons of 5-11 μm width and several hundreds of micrometers length were observed for the first time (FIG. 4). By increasing the solution pH, ribbon formation slowed, decreased in yield, increased in helicity and entanglements of the giant ribbons. An interdigitated lamellar packing model of acidic SL-COOH molecules with a long period of 2.78 nm, stabilized by both the strong hydrophobic association between cis-9-octadecenoic chains and strong disaccharide-disaccharide hydrogen bonding was proposed (FIG. 5). New sophorolipid structures will be useful to manipulate the tendency to self assemble as well as the range of useful structures formed.
Various sophorolipid-based structures have been found useful as therapeutic agents. Experimental studies in mice and rats were performed to assess effects of sophorolipids on sepsis-related mortality when administered as a (1) single bolus versus sequential dosing and (2) natural mixture versus individual derivatives compared with vehicle alone (Bluth et al., 2006; Hardin et al., 2007). Intra-abdominal sepsis was induced in male, Sprague Dawley rats, 200 to 240 g, via cecal ligation and puncture. Sophorolipids (5-750 mg/kg) or vehicle (ethanol/sucrose/physiological saline) were injected intravenously (i.v.) via tail vein or inferior vena cava at the end of the operation either as a single dose or sequentially (q24×3 doses). The natural mixture was compared with select sophorolipid derivatives (n=10-15 per group). Sham-operated animals served as non-sepsis controls. Survival rates were compared at 1 through 6 day post sepsis induction. The results showed sophorolipid treatment at 5 mg/kg body weight improved survival in rats with cecal ligation and puncture-induced septic shock by 28% at 24 h and 42% at 72 h for single dose, 39% at 24 h and 26% at 72 h for sequential doses, and 23% overall survival for select sophorolipid derivatives when compared with vehicle control (P<0.05 for sequential dosing) (FIG. 6). Toxicity was evident and dose-dependent with very high doses of sophorolipid (375-750 mg/kg body weight) with histopathology demonstrating interstitial and intra-alveolar edema with areas of micro-hemorrhage in pulmonary tissue when compared with vehicle controls (P<0.05). No mortality was observed in sham operated controls at all doses tested. Therefore, it was concluded that administration of sophorolipids after induction of intra-abdominal sepsis improves survival. The demonstration that sophorolipids can reduce sepsis-related mortality with different dosing regimens and derivatives provides continuing evidence toward a promising new therapy. New sophorolipid structures may be useful to improve their potency for treatment of sepsis.
Other work has shown that modified sophorolipids have antibacterial, antiviral, and anti-inflammatory properties (Mueller et al, 2006; Shah et al., 2005). In one example, sophorolipids were shown to down-regulate expression of proinflammatory cytokines including interleukin (Hagler et al. 2007). Furthermore, Table 1 below shows how antibacterial properties of sophorolipids can be increased by up to 1000 times relative to the natural SL mixture by simple modifications of sophorolipids such as esterification of fatty acid carboxyl groups and selective acetylation of disaccharide hydroxyl groups. Therefore, those skilled in the art will recognize that the changes in sophorolipid structure described herein can be used to improve sophorolipid-based therapeutics so they are more potent, less toxic, and have other desirable characteristics.
TABLE 1NaturalSL-E-9SL-A-4SL-E-4SL-E-5SL-E-1SLMIC100MIC100MIC100MIC100MIC100MIC100Eserichia coli1.675551.675Moraxella1.6752.05 × 10−26.17 × 10−26.17 × 10−25Ralstoniaeutropha555555RhodoccocuserythropolisN/A0.566.86 × 10−3555Salmonella choleraesuis55551.675Note:All values in Table 1 are mg/ml. “Natural SL” refers to the mixture of acidic and lactonic sophorolipids obtained from fermenation. Strucures of sophorolipids are shown in FIG. 13.
Sophorolipids were shown to be useful for protection against human immunodeficiency virus (HIV) and as a vaginal topical microbicide (Shah et al, 2005). Thus far with the limited range of sophorolipid derivatives available, the sophorolipid diacetate ethyl ester derivative was found to be the most potent spermicidal and virucidal agent. Its virucidal activity against HIV and sperm-immobilizing activity against human semen are similar to those of nonoxynol-9. However, it also induces sufficient vaginal cell toxicity to raise concerns about its applicability for long-term microbicidal contraception. Therefore, those skilled in the art will recognize that the changes in sophorolipid structure described herein can be used to improve virucidal activity against HIV and sperm-immobilizing activity against human semen. Furthermore, new variants may reduce vaginal cell toxicity.
Sophorolipids were also found to mediate cytotoxic responses to pancreatic cancer cell lines (Fu et al., 2008). These anticancer responses were dose- and derivative-dependent and likely kill cancer cells by necrosis (FIG. 7 illustrates SL structure-activity relationships with HPAC cells). Furthermore, these agents are specific to cancer cells in that they did not affect normal human cells. Hence, sophorolipids represent a unique and novel class of drugs. New sophorolipid derivatives may be useful to increase their potency and specificity against pancreatic and other cancer types. Therefore, those skilled in the art will recognize that the changes in sophorolipid structure described herein can be used to improve the potency and specificity of sophorolipids against pancreatic and other cancer types.